Members of the Hedgehog (“hh ”) family of signaling molecules mediate many important short-and long range patterning processes during invertebrate and vertebrate development.
To date, the combined screening of mouse genomic and cDNA libraries has identified three mammalian hh counterparts referred to as Desert hedgehog (Dhh), Sonic hedgehog (Shh) and Indian hedgehog (Ihh), which also exist in other mammals as well as in fish and birds. Other members include Moonrat hedgehog (Mhh), as well as chicken Sonic hh and zebrafish Sonic hh. Mouse and chicken Shh and mouse Ihh genes encode glycoproteins which undergo cleavage, yielding an amino terminal fragment of about 20 kDa and a carboxy terminal fragment of about 25 kDa. The 20 kDa fragment has the consensus sequence SEQ ID NO: 1 (FIG. 5). Publications disclosing these sequences, as well as their chemical and physical properties, include Hall et al., (1995) Nature 378, 212-216; Ekker et al., (1995) Current Biology 5, 944-955; Fan et al., (1995) Cell 81, 457-465; Chang et al., (1994) Development 120, 3339-3353; Echelard et al., (1993) Cell 75, 1414-1430; and PCT Patent Application WO 9523223 (Jessell, Dodd, Roelink and Edlund).
Human Shh is synthesized as a 45 kDa precursor protein that is also autocatalytically cleaved to yield: (I) a 20 kDa N-terminal fragment that is responsible for all known hedgehog signaling activity; and (II) a 25 kDa C-terminal fragment that contains the autoprocessing activity (Lee, J. J., et al. (1994) Science 266, 1528-1536; Bumcrot, D. A., et al. (1995), Mol. Cell Biol. 15, 2294-2303; Porter, J. A., et al. (1995) Nature 374, 363-366).
The N-terminal fragment consists of amino acid residues 24-197 of the full-length precursor sequence. The N-terminal fragment remains membrane-associated through the addition of a cholesterol at its C-terminus (Porter, J. A., et al. (1996) Science 274,255-258; Porter, J. A., et al. (1995) Cell 86,21-34). This cholesterol is critical for restricting the tissue localization of the hedgehog signal. The addition of the cholesterol is catalyzed by the C-terminal domain during the processing step. As a result of the membrane tethering, a high local concentration of N-terminal hedgehog peptide is generated on the surface of the hedgehog producing cells.
The hedgehog proteins regulate various aspects of embryonic development both in vertebrates and invertebrates (for reviews see Perrimon, N. (1995) Cell 80, 517-520 and Johnson, R. L., and Tabin, C. (1995) Cell 81, 313-316). The most well-characterized hedgehog protein is Sonic hedgehog (Shh), involved in anterior-posterior patterning, formation of an apical ectodermal ridge, hindgut mesoderm, spinal column, distal limb, rib development, and lung development, and inducing ventral cell types in the spinal cord, hindbrain and forebrain (3-8). While the mechanism of action of hedgehog proteins is not fully understood, the most recent biochemical and genetic data suggest that the receptor for Shh is the product of the tumor suppressor gene, patched (Marigo, V., et al. (1996) Nature 384, 176-179; Stone, D. M., et al. (1996) Nature 384, 129-134) and that other proteins; smoothened (Stone, D. M., et al. (1996) Nature 384, 129-134; Alcedo, J., et al. (1996) Cell 86, 221-232), Cubitus interruptus (Dominguez, M., et al. (1996) Science 272, 1621-1625; Alexandre, C., et al. (1996) Genes & Dev. 10, 2003-2013), andfused (Therond, P. P., et al. (1996) Proc. Natl. Acad. Sci. USA 93, 4224-4228) are involved in the hedgehog signaling pathway. The interaction of a hedgehog protein with one of its cognate receptors, patched (ptc), sets in motion a cascade involving the activation and inhibition of downstream effectors, the ultimate consequence of which is, in some instances, a detectable change in the transcription or translation of a gene. Hedgehog and its cognate receptor patched (ptc) are expressed in the epithelial and/or mesenchymal cell components of the skin (i.e., the hair follicle). See Parisi et al., (1998) Cell Res 8, 15-21; St. Jacques et al., (1998) Current Biology, 8, 1058-1068; and Dahmane et al., (1997) Nature, 389, 876-880. The two-way interaction between epithelial and the dermal mesenchymal cells directs the subsequent development of hair follicles. Disrupting this interaction might lead to a modulation of proliferation and/or differentiation events that give rise to hair and/or epithelial tissue structures such as the gut.